Targeting IRE1α improves insulin sensitivity and thermogenesis and suppresses metabolically active adipose tissue macrophages in obesity

Overnutrition engenders the expansion of adipose tissue and the accumulation of immune cells, in particular, macrophages, in the adipose tissue, leading to chronic low-grade inflammation and insulin resistance. In obesity, several proinflammatory subpopulations of adipose tissue macrophages (ATMs) identified hitherto include the conventional “M1-like” CD11C-expressing ATM and the newly discovered metabolically activated CD9-expressing ATM; however, the relationship among ATM subpopulations is unclear. The ER stress sensor inositol-requiring enzyme 1α (IRE1α) is activated in the adipocytes and immune cells under obesity. It is unknown whether targeting IRE1α is capable of reversing insulin resistance and obesity and modulating the metabolically activated ATMs. We report that pharmacological inhibition of IRE1α RNase significantly ameliorates insulin resistance and glucose intolerance in diet-induced obesity mice. IRE1α inhibition also increases thermogenesis and energy expenditure, and hence protects against high fat diet-induced obesity. Our study shows that the “M1-like” CD11c+ ATMs are largely overlapping with but yet non-identical to CD9+ ATMs in obese white adipose tissue. Notably, IRE1α inhibition diminishes the accumulation of obesity-induced metabolically activated ATMs and “M1-like” ATMs, resulting in the curtailment of adipose inflammation and ensuing reactivation of thermogenesis, without augmentation of the alternatively activated M2 macrophage population. Our findings suggest the potential of targeting IRE1α for the therapeutic treatment of insulin resistance and obesity.

Flow cytometry analysis of ATMs from SVFs of the eWAT from 20-week HFD-fed mice.SVFs were cultured and treated with 0.01% DMSO control or STF (30 µM) for 20 h followed by staining and flow analysis, gated against CD9 antibody (A) or CD11C antibody (B).The gating path followed the one as Supplemental Figure 5A.C. mRNA levels of indicated genes in CD11C + ATMs sorted from SVFs cultured and treated with DMSO or STF, assessed by qRT-PCR.D-E.Comparison of mRNA levels of indicated proinflammatory genes between CD11C + and CD11C -ATMs (D) and between CD9 + and CD9 -ATMs (E), sorted from SVFs, assessed by qRT-PCR.F-G. mRNA levels of indicated proinflammatory genes (F) and spliced-XBP1 (G) in CD9 -ATMs sorted from SVFs cultured and treated with DMSO or STF, assessed by qRT-PCR.F-G. mRNA levels of indicated proinflammatory genes (F) and spliced-XBP1 (G) in CD11C - ATMs sorted from SVFs cultured and treated with DMSO or STF, assessed by qRT-PCR.Data were expressed as mean ± SEM and analyzed using the unpaired two-tailed Student's t-test between two samples or ANOVA with multiple comparisons.* P < 0.05, * * P < 0.01, and * * * P < 0.001..  Table S1a Table S1b Table S1c Table S1 Table S1.Effect of STF treatment on "M1-like" and "M2-like" ATMs in obesity.

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The total cell number and density of populations from CD11C + CD206 -ATMs in the eWATs of mice with ND, HFD-Veh, or HFD-STF (a), CD11C -CD206 -ATMs in the eWATs of mice with ND, HFD-Veh, or HFD-STF (b), and F4/80      3 Primer sequences used in this work

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stress and IRE1α activation in adipose tissues in DIO mice.A-B. mRNA levels of ER stress genes (A) and of IRE1α RIDD target genes (B) in the eWAT from mice fed ND (normal diet) and HFD for 14 weeks, assessed by qRT-PCR.The results are expressed as fold change and are representative of 3 independent experiments.C. Glucose tolerance test performed on mice on ND and HFD for 8 weeks.Blood glucose levels measured at indicated time points after intraperitoneal injection of glucose (1.5 g/kg body weight) following 6-h fasting.D-D'.XBP1 mRNA levels were analyzed from eWAT of DIO mice treated with vehicle or STF by RT-PCR and resolved by agarose gel electrophoresis.The full length (unspliced, XBP1-u) and spliced (XBP1-s) forms of XBP1 mRNA were indicated (D) and quantified (D').GAPDH mRNA was used as an internal control.The data shown are representative of 3 independent experiments.E. mRNA levels for indicated genes were analyzed in eWAT of DIO mice treated with vehicle or STF by qRT-PCR.The results are expressed as fold change and are representative of 3 independent experiments.Data are expressed as mean ± SEM. * P < 0.05, * * P < 0.01, and * * * P < 0.001.protects against diet-induced obesity and promotes energy expenditure.A. Body weight of DIO mice treated with vehicle (n=5) or STF (n=8) before and after treatment for 4 weeks.B. Daily food intake from DIO mice treated with vehicle or STF, measured for three consecutive days.C-D.Adipocyte area per cell in the sections of eWAT (C) and iWAT (D).E-E'.Curves (E) and quantification (E') of oxygen consumption levels.F-F'.Curves (F) and quantification (F') of CO2 production levels.G-G'.Curves (G) and quantification (G') of energy expenditure levels calculated.For E-G', the data was normalized by body weight for analysis for group effect.Data were represented as mean ± SEM and analyzed using the unpaired two-tailed Student's t-test between two samples or ANOVA with multiple comparisons .* P < 0.05, * * P < 0.01, and * * * P < 0.001.promotes thermogenesis in WAT of DIO mice. A. Quantification of immune-signal intensity of UCP1 in eWAT sections from ND mice and DIO mice treated with vehicle or STF.B-C.UCP1 immunofluorescence staining of iWAT sections from ND-fed mice and DIO mice treated with vehicle or STF.Representative images of UCP1 immunofluorescence staining of iWAT (B) and quantification of immune-signal intensity of UCP1 (C).D. mRNA levels of thermogenic genes in iWAT from DIO mice treated with vehicle or STF, assessed by qRT-PCR.The results are expressed as fold change and are representative of 3 independent experiments.E-F. mRNA levels of b-adrenoceptor genes in eWAT (E) and iWAT (F) from DIO mice treated with vehicle or STF, assessed by qRT-PCR.The results are expressed as fold change and are representative of 3 independent experiments.Data are the mean ± SEM. * P < 0.05, * * P < 0.01, and * * * P < 0.001.inhibits IRE1α activity in the SVF of obese eWAT. A. Percentage of CLS area in iWAT sections from DIO mice treated with vehicle or STF.B-C.Splicing of XBP1 mRNA was analyzed from the SVFs of eWAT of DIO mice treated with vehicle or STF by RT-PCR and resolved by agarose gel electrophoresis.The full length (unspliced, XBP1-u) and spliced (XBP1-s) forms of XBP1 mRNA were indicated (B) and quantified with t-XBP1 as the sum of XBP1-u and XBP1-s (C).GAPDH mRNA was used as an internal control.The data shown are representative of 3 independent experiments.D. mRNA levels for indicated genes were analyzed in the SVFs of eWAT of DIO mice treated with vehicle or STF by qRT-PCR.The results are expressed as fold change and are representative of 3 independent experiments.Data are the mean ± SEM. * P < 0.05, * * P < 0.01, and * * * P < 0.001.Figure 5. STF suppresses obesity-driven ATM accumulation and adipose inflammation.A. Representative pseudocolor cell density flow cytometry plots showing the gating strategy used for analysis of macrophage subsets (F4/80 + CD11b + ) of SVFs of eWAT from ND-fed mice and DIO mice treated vehicle or STF, for figures shown in Fig. 5E.For Fig. 5F, G, I, J, Fig. 6H-N and Fig. 7G, markers were further gated immediately following the steps shown here.Numbers indicate percentage of gated cells.B. Flow cytometry analysis of CD206 signal in CD11c + ATMs (F4/80 + CD11b + CD11C + ) from eWAT SVFs of mice with an overlay of gated CD206 marker for ND and HFD.Complete gating path follows the strategy shown in A. C. mRNA levels of indicated proinflammatory cytokine/chemokine genes in the SVFs of iWAT of DIO mice treated with vehicle or STF, assessed by qRT-PCR.The results are expressed as fold change and are representative of 3 independent experiments.D. mRNA levels of splice XBP1 in the CD11C + ATMs (F4/80 + CD11b + CD11C + ) sorted from SVFs of eWAT of DIO mice treated with vehicle or STF, assessed by qRT-PCR.The results are expressed as fold change and are representative of 3 independent experiments.Data are the mean ± SEM. * P < 0.05, * * P < 0.01, and * * * P < 0.001.Suppl.Fig. 5 (cont.)suppresses metabolically active ATMs in DIO mice.A-C. mRNA levels of indicated genes in the SVFs of iWAT from ND, HFD-veh and HFD-STF mice, assessed by qRT-PCR.The results are expressed as fold change and are representative of 3 independent experiments.D. Representative images of immunofluorescent staining of CD9 (red) in the eWAT sections of ND-and HFD-fed mice.DAPI (blue) for nuclear staining.E. Representative images of immunofluorescent staining of Trem2 (red) in the eWAT sections of ND-and HFD-fed mice.DAPI (blue) for nuclear staining.F. Flow cytometry analysis of CD63 signal in CD9 + ATMs from eWAT SVFs of DIO mice, gated against CD9 and CD63 antibodies sequentially following the gating strategy shown in Supplemental Figure 5A.G. Flow cytometry analysis of CD9 + and CD63 + ATMs from eWAT SVFs of ND, HFD-Veh and HFD-STF mice, gated against CD9 and Trem2 antibodies simultaneously following the gating strategy shown in Supplemental Figure 5A.H. Flow cytometry analyses of Trem2 signal in CD9 + ATMs (left panel) and of CD9 signal in Trem2 + ATMs (right panel) from eWAT SVFs of DIO mice, gated against CD9 and Trem2 antibodies sequentially (left panel) or against Trem2 and CD9 sequentially (right panel) following the gating strategy shown in Supplemental Figure 5A.Data are the mean ± SEM. * P < 0.05, * * P < 0.01, and * * * P < 0.001.Suppl.Fig. 6 cont.S Figure 7. STF suppresses CD9+ and Trem2+ ATMs in DIO mice. A. Representative images of immunofluorescent staining of CD9 (red) and Trem2 (green) in the BAT sections of HFD-fed mice treated with Veh or STF.DAPI (blue) for nuclear staining.B-C.Representative images of immunofluorescent staining in the iWAT sections of HFD-fed mice treated with Veh or STF (B) and quantification (C).CD9 (red), Trem2 (green), and DAPI for nuclear staining (blue).Suppl.Fig.7.New

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suppresses pro-inflammatory cytokines genes expression in ATM in vitro.A-B.

Table S2 . Characterization of and the effect of STF treatment on CD9 + ATMs in DIO mice.
The percentage, total cell number, and density of populations from CD9 + CD63 + ATMs in the eWATs of mice with ND, HFD-Veh, or HFD-STF.Data were obtained from 2 batches of 4-5 mice each and are the mean ± SEM. * P < 0.05, * * P < 0.01, and * * * P < 0.001